Cas9 (CRISPR-associated protein 9) is a DNA cutting endonuclease that is part of the CRISPR immune system in bacteria and archaea. It has been adapted for use as a CRISPR genome editing tool. The CRISPR-Cas9 system is widely used in genetic engineering, medicine, agriculture, and biotechnology.
The Cas9 endonuclease introduces site-specific double-stranded breaks in DNA, enabling gene inactivation, removal, or insertion through cellular repair mechanisms such as non-homologous end joining or homologous recombination.
Cas9 was discovered in Streptococcus pyogenes and later in S. thermophilus. It functions with two small RNAs—crRNA and tracrRNA—to form a four-component complex. In 2012, Doudna and Charpentier fused the RNAs into a single-guide RNA, enabling Cas9 to cut specific DNA sequences. These developments spurred genome modification efforts, including groups led by Zhang and Church who showed genome editing in human cells.
Variants of Cas9, including Cas9 nickase (Cas9n), that create single-strand breaks, and versions recognizing different PAM sequences, address specific Cas9 limitations.
CRISPR-Cas9 technology has raised ethical and regulatory concerns, particularly regarding human germline editing and genetic enhancement.
History Excerpt end
Development of Cas9 as a genome editing tool
Cas9 was first identified in Streptococcus pyogenes as a CRISPR-associated protein, originally referred to as Cas5, involved in the bacterial adaptive immune system. Studies found Cas9 contains a nuclease domain, the effector enzyme responsible for generating double-stranded DNA breaks in the type II CRISPR system.
Researchers identified that Cas9 activity depends on two RNA molecules: CRISPR RNA (crRNA), which guides the enzyme to target DNA sequences, and trans-activating CRISPR RNA (tracrRNA), which is required for nuclease activity.
In 2012, Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a simplified two-component system by fusing the two RNA molecules into a single-guide RNA that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. That same year, research showed that CRISPR Cas9 components could be programmed for site-specific double stranded DNA breaks in vitro, laying the foundation for genome editing.
By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target and cleave DNA sequence for separation. Around the same time, a collaboration including Virginijus Šikšnys, Gasiūnas, Barrangou, and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed by modifying its crRNA sequence to target specific DNA sites.
In 2013, research groups led by Feng Zhang and George Church reported successful targeted DNA editing in human genomes, marking the first use of CRISPR-Cas9 for targeted modification in eukaryotic systems. These findings advanced the development of CRISPR-Cas9 as a versatile genome editing tool across a wide range of organisms.
Beyond DNA cleavage, CRISPR Cas9 has been modified to function as programmable transcription factors that allow activation or silencing of targeted genes. The significance of the system's development was formally recognized in 2020, when Emmanuelle Charpentier and Jennifer Doudna were awarded a Nobel Prize in Chemistry for their work in CRISPR Cas9 genome editing.
Mechanism Native Cas9 is a 160-kilodalton endonuclease that requires a guide RNA composed of two distinct RNA molecules: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
Research
2013 In 2013, Mali, et al. and Gilbert, et al. reported that non-cleaving Cas9 variants can control transcriptional activators or repressors at specific DNA sequences. In the same year, Bikard, et al. reported that dCas9 can block RNA polymerase at promoters or during elongation, silencing genes.
2014 In 2014, Esvelt, Smidler, Catteruccia, and Church suggested that Cas9-based gene drives could spread genetic modifications through entire organism populations.
2017-19 In 2017, Li, et al. reported that Cas9 can target the end of the Hepatitis B viral genome ends, reducing infection.
Jensen et al., reported that dCas9 alters chromatin structure when fused to chromatin remodelers, . O'Geen, et al., reported that dCas9 with FOG1 represses genes by methylating H3K27.
Lowder, et al., reported that dCas9 represses plant genes like AtCSTF64 when fused to repressor domains.
In 2018, Lee, et al., reported that engineered Cas9 reduces off-target effects by altering reaction rates.
In 2019, Chen and Page-McCaw reported that Cas9 suppresses HIV-1 long terminal repeats, mutating the viral genome.
2020 In 2020, Uddin, Rudin, and Sen reported that Cas9 variants, such as Cas9 nickase, improve genome editing by reducing off-target effects.
2025 In 2025, Zhou, Diao, Li, and colleagues reported that apoNmeCas9 (an RNA-free form of NmeCas9) can stimulate Cas1–Cas2-mediated CRISPR spacer acquisition in a type II-C system when CRISPR RNA levels are low.
CRISPR-mediated immunity CRISPR systems in bacteria and archaea function as programmable restriction enzymes to combat viruses and plasmids. CRISPR loci consist of short, palindromic repeats and variable spacers (24–48 nucleotides) that store sequences from foreign DNA. Adjacent cas genes, including universal cas1 and cas2, and type II-specific cas9, encode proteins for immunity. Cas9 uses crRNA and tracrRNA to form a complex that targets and cleaves foreign DNA matching the spacer sequence.
Adaptation During the adaptation stage, CRISPR systems integrate spacers from viral"protospacers" into the CRISPR locus. It requiring the presence of a PAM, such as NGG in S.pyogenes) for recognition. Cas9 ensures functional spacer acquisition. However, spacers can be lost via homologous recombination.
ApoCas9 has been reported to stimulate spacer acquisition in type II-C systems and to couple acquisition activity to CRISPR RNA abundance with low crRNA (e.g.: in cells undergoing array neogenesis or natural array contraction) associated with increased acquisition and higher crRNA (expanded arrays) associated with reduced acquisition. The nuclease lobe alone is sufficient to stimulate acquisition, whereas crRNA-dependent regulation required full-length Cas9. This activity is evolutionarily conserved across several type II-C Cas9 orthologues.
CRISPR processing RNA polymerase transcribes the CRISPR locus into pre-crRNA, which is processed by specific endoribonucleases into mature crRNAs, each containing one spacer and a partial repeat.
Interference In the interference stage, the crRNA guides Cas9 together with tracrRNA to complementary foreign DNA sequences. If the targer sequence matches the spacer and a PAM is present, Cas9 introduces double-stranded breaks in DNA, stoping viral replication.
Transcription regulation Inactive Cas9 (dCas9), lacking endonuclease activity, can bind DNA and regulate transcription. In eukaryotic cells, dCas9 targets enhancer sequences to prevent transcription factor binding and gene expression. Genome-wide screens using dCas9-based repression have been conducted to systematically identify gene funciton.
In addition to DNA targeting, Cas9 has been shown to cleave mRNA when hybridized with complementary single-stranded containing a PAM complement. When fused to transcription activators, dCas9 can be used to activate genes(CRISPRa).
Structural studies Cas9 has a bi-lobed structure with an alpha-helical lobe (REC) and a nuclease lobe (NUC) containing HNH and RuvC domains, connected by a bridge helix. The nuclease lobe contains HNH domains which cleaves target DNA strands, and RuvC which cleaves the non-target strand. The PAM-interacting domain recognizes the NGG PAM sequence. The single-guide RNA (sgRNA), replaces the crRNA-tracrRNA complex, and forms a T-shaped structure with the target DNA. A 2014 study reported that REC1 and bridge helix domains are critical for sgRNA recognition.
Challenges in bacterial editing Bacterial restriction-modification systems can degrade foreign DNA introduced into the cell. These systems recognize specific DNA sequences and cleave foreign DNA, including Cas9-introduced genes, hindering genome editing.
Applications
Gene editing Cas9 edits genomes in a range of species, including yeast (Saccharomyces cerevisiae), Candida albicans, zebrafish (Danio rerio), fruit flies (Drosophila melanogaster), ants (Harpegnathos saltator and Ooceraea biroi), mosquitoes (Aedes aegypti), nematodes (Caenorhabditis elegans), plants, mice (Mus musculus domesticus), and monkeys.
Cellular defense CRISPR/Cas9 can help with bacterial defense against invaders such as bacteriophages, plasmids, and DNA viruses. Cas9, guided by RNA, acts as an endonuclease enzyme, targeting and cleaving foreign DNA. It unwinds foreign DNA and cleaves matching sites, similar to the RNA interference mechanism in eukaryotes.
Gene regulation Other versions of Cas9 can bind but not cleave DNA. They can be used to control transcriptional activator or repressor genes and manage gene activation and repression.
More technically, Cas9 is a RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system in Streptococcus pyogenes. S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 nucleotide spacer region of the guide RNA (gRNA). If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism parallels the RNA interference (RNAi) mechanism in eukaryotes. Apart from its original function in bacterial immunity, Cas9 has been utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in model organisms. Research on cas9 variants has been a promising way of overcoming the limitation of the CRISPR-Cas9 genome editing. Examples include Cas9 nickase (Cas9n), a variant that induces single-stranded breaks (SSBs) or variants recognizing different PAM sequences. Alongside zinc finger nucleases and transcription activator-like effector nuclease (TALEN) proteins, Cas9 is becoming a prominent tool in the field of genome editing.
Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and zinc fingers), engineering Cas9 to target new DNA is straightforward. Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA (chiRNA). Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms. In 2015, Cas9 was used to modify the genome of human embryos for the first time.
CRISPR-mediated immunity